The Journal of Physiology ain .Kostrominova Y. Tatiana 1 Ramaswamy S. Krishnan rats old very muscles and skeletal mice in dystrophic impaired of is force of transmission Lateral Physiol J eia col n the and School, Medical C eatet fBoeia Engineering, Biomedical of Departments 01TeAtos ora compilation Journal Authors. The 2011 8. 21)p 1195–1208 pp (2011) 589.5 D,etno iiou longus. digitorum extensor EDL, 7732C [email protected] School, Email: Medical Abbreviations Michigan USA. of 48109-0622, MI University Arbor, Physiology, Ann Integrative St, [email protected] Catherine and Email: E Molecular 1301 USA. MSII, of 48109-2002, Department MI Arbor, Michele: Ann E. Place, Pitcher D. Zina 109 BSRB, 2035 School, Medical and humans dystrophic In matrix. extracellular the to cytoskeleton fibre Abstract to lead DGC the injury. of contraction-induced disruptions and and force instability of transmission lateral the were for intact essential the muscles an is muscles, which DGC skeletal of in in contractions rats, during forces that old conclude We severely. impaired very of transmitted were forces laterally and the transmission mice disrupted, was dystrophic fibres lateral of of (DGC) muscles complex and glycoprotein that skeletal -associated for longitudinal demonstrated contrast, then rats In We different. muscles forces. and not parallel-fibred of mice whole, transmission of wild-type lateral surface the for the of to measurements attached permitted that and developed mammals, was in phenomenon apparatus this demonstrate To ‘yoke’ loss. no a or little with surface muscle the to laterally summary Non-technical Rsbitd1 oebr21;acpe fe eiin4Jnay21;fis ulse nie1 aur 2011) January 10 online published first 2011; January authors 4 Corresponding revision after accepted 2010; November 17 (Resubmitted now We (WT force. wild-type lateral young in of of measurement demonstrated muscles epimysium the in the enabled been surface to and that not tendons attached report muscle the that has the between developed midway force to was muscles apparatus of fibre of ‘yoke’ activated transmission unique fibres. A an lateral muscle muscles. from to but mammalian laterally damage attenuation, severe force without causing of DGC occurs transmission the muscles, of frog structure the In disrupt gene dystrophin the marltrltasiso ffrecuigisaiiyadicesdssetblt ffirsto fibres of susceptibility increased and In instability decrement. causing without injury. force function DGC contraction-induced and of structure the DGC transmission through in disruptions lateral fibre animals, old impair to mice very or WT fibre dystrophic young of from muscles of in laterally contrast, muscles transmitted skeletal are by developed rats forces and contractions, during that conclude for We contrast, In decrement. both no of or little showed of laterally muscles transmitted forces forces, longitudinal 5 mdx colo ieilg,a h nvriyo ihgn n ro,Mcia 80-20 USA 48109-2200, Michigan Arbor, Ann Michigan, of University the at Kinesiology, of School h ytohngyorti ope DC rvdsa seta ikfo h muscle the from link essential an provides (DGC) complex dystrophin–glycoprotein The mdx T,atro iils G,dsrpi-soitdgyorti ope;EM xrclua matrix; extracellular ECM, complex; glycoprotein dystrophin-associated DGC, tibialis; anterior ATB, ieadvr l assoe ao eutosi h xrsino dystrophin. of expression the in reductions major showed rats old very and mice 3 ieadvr l as ocstasitdltrlywr mardsvrl.Muscles severely. impaired were laterally transmitted forces rats, old very and mice 1 ailE Michele E. Daniel , akL Palmer L. Mark , C .A alnr eateto oeua n nertv hsooy nvriyo Michigan of University Physiology, Integrative and Molecular of Department Faulkner: A. J. 01TePyilgclSceyDI 10.1113/jphysiol.2010.201921 DOI: Society Physiological The 2011 2 ugr,Scino lsi Surgery, Plastic of Section Surgery, h oc eeoe yasnl bei rgmslsi transmitted is muscles frog in fibre single a by developed force The 1 , 5 3 akH a e Meulen der van H. Jack , , 4 n onA Faulkner A. John and ) ieadrt,cmae vrawd ag of range wide a over compared rats, and mice 3 oeua n nertv hsooy and Physiology, Integrative and Molecular 1 2 , 3 ,AbigailRenoux mdx ie uain in mutations mice, 3 , 4 nenlMdcn nthe in Medicine Internal 1195 1196 K. S. Ramaswamy and others J Physiol 589.5

Introduction skeletal muscles lack the DGC, even repeated isometric contractions cause a severe contraction-induced injury A major breakthrough occurred in the understanding of (Claflin & Brooks, 2008) and lengthening contractions muscle mechanics when Street (1983) provided the first cause an injury that is even more severe (DelloRusso physiological evidence that confirmed, in experiments on et al. 2001; Li et al. 2006). The conclusion that in vitro semitendinosus muscles of frogs, that two pathways dystrophin and the DGC protect fibres of force transmission existed, one longitudinal and the from contraction-induced injury is supported by the other lateral. These experiments demonstrated for the protection from contraction-induced injury provided the first time that most, if not all of the force developed skeletal muscles of dystrophic mice through the expression by a single muscle fibre longitudinally was transmitted of a mini-dystrophin fusion gene that restored dystrophin laterally through the adjacent extracellular matrix (ECM) expression and DGC function in skeletal muscles (Li et al. and muscle fibres to the epimysium of the skeletal muscle. 2006). Despite this early demonstration of the existence of the Unlike muscular dystrophy, with a single underlying lateral transmission of force in skeletal muscles of frogs, cause arising from the loss of dystrophin, the age-related the inability to perform similar experiments on any changes in skeletal muscles arise from multiple underlying mammalian skeletal muscle resulted in a complete lack causes that are largely unknown. The age-related structural of direct evidence that this phenomenon also occurred and functional deficits that have been demonstrated in in the skeletal muscles of mammalian species. Although skeletal muscles of mice (Brooks & Faulkner, 1988), rats no measurements have been made that demonstrate (Larsson et al. 1991) and humans (Dedrick & Clarkson, lateral transmission of force in mammalian muscles, many 1990; Frontera et al. 1991; Ploutz-Snyder et al. 2001) investigators (Pardo et al. 1983; Ervasti & Campbell, 1993; include a loss of motor units (Doherty et al. 1993); muscle Worton, 1995; Rybakova et al. 2000; Paul et al. 2002; atrophy; fatigability and weakness (Young et al. 1984, 1985; Bloch & Gonzalez-Serratos, 2003; Campbell & Stull, 2003; Brooks & Faulkner, 1988; Frontera et al. 1991); a decrease Ervasti, 2003; Huijing, 2003; Bloch et al. 2004; Abmayr & in maximum and sustained power (Faulkner et al. 2008); Chamberlain, 2006; Anastasi et al. 2008; Claflin & Brooks, and an increased susceptibility to contraction-induced 2008) have assumed that the lateral transmission of force injury (Dedrick & Clarkson, 1990; DelloRusso et al. 2001; must function as effectively in mammalian muscles, as Ploutz-Snyder et al. 2001; Li et al. 2006). The possibility evidenced in the muscles of frogs (Street, 1983), despite that during the ageing of mammalian skeletal muscles, theabsenceofanydirectevidencethatsuchisthecase. the loss of dystrophin expression leads to an impaired The pathway proposed by these investigators for lateral transmission of force has not been investigated pre- the lateral transmission of force has focused primarily viously. Here we demonstrate for the first time that force is on the potential of , first described by transferred laterally without decrement in skeletal muscles Pardo and colleagues (1983), to provide the necessary of young wild-type (WT) mice and rats. In contrast, in linkage for the transfer of force laterally from the skeletal muscles of both mdx mice and very old rats, z-discs of skeletal muscle fibres to the ECM (Ervasti, the lateral transmission of force is impaired severely. 2003; Bloch et al. 2004). Within striated skeletal Throughout the manuscript the terms WT, mdx and very muscle fibres, the dystrophin-associated glycoprotein old muscles will be used when appropriate, rather than complex (DGC), situated primarily within costameres, designating the species. The observations on WT, mdx and appears to provide the necessary connection between very old muscles suggest that disruptions associated with the force-generating structures, the , laterally muscular dystrophy, or acquired disruptions of the DGC through the and basement membrane into associated with ageing, may interfere with the mechanical the ECM that is shared with the surrounding muscle connections between skeletal muscle fibres and the ECM fibres (Ervasti & Campbell, 1991; Worton, 1995; Henry & and that such disruptions may lead to impairments in the Campbell, 1996; Bloch & Gonzalez-Serratos, 2003; Ervasti, lateral transmission of force and the subsequent muscle 2003; Michele & Campbell, 2003; Bloch et al. 2004; Lapidos dysfunctions that are associated with both dystrophy and et al. 2004; Anastasi et al. 2008). During contractions, ageing. these potential pathways for the lateral transmission of forces provide the possibility of stabilizing the lengths of sarcomeres that vary in their capability of developing Methods force (Macpherson et al. 1997; Panchangam et al. 2008). The DGC appears to be essential in mammalian skeletal To investigate the effect of the lack of dystrophin on muscles, since a loss of its components causes muscular the lateral transmission of force, adult (12–15 months of dystrophy in both humans (Hoffman et al. 1987; Worton, age) WT (n = 6) and mdx (n = 6) male mice from the 1995; Bloch et al. 2004) and mice (Rybakova et al. C57BL/10ScSn-mdx/J strain were purchased from The 2000; Li et al. 2006). In dystrophic (mdx)mice,whose Jackson Laboratory (Bar Harbor, ME, USA). In addition,

C 2011 The Authors. Journal compilation C 2011 The Physiological Society J Physiol 589.5 Lateral transmission of force in skeletal muscles of mice and rats 1197 to determine the age-related influences, F344 × Brown moist by periodic applications of isotonic saline. For a Norway F1 young rats of3monthsofage(n = 6), old given preparation of either muscle, single twitches of the rats of 30–33 months of age (n = 6), and very old rats of whole muscle were initiated by square wave pulses of 36–38 months of age were obtained from the National 0.2 ms duration administered to the appropriate motor Institutes of Aging. The inclusion of the two ‘old age’ nerve. The stimulation voltage and muscle length (Lo) groups was based on the linear decline in the skeletal were adjusted to produce the maximum isometric twitch muscles of humans of the number of motor units (Doherty force (Pt). With the muscle at Lo, tetanic contractions were et al. 1993) and the number of fibres in skeletal muscles produced by the 0.2 ms square wave pulses applied for (Lexell et al. 1988) that result in a linear decline in strength, 300 ms at a frequency of 40 Hz. Subsequent tetanic contra- power, maximum oxygen uptake and consequently in the ctions were produced with increments of 30 Hz in the records for running and weight lifting (Faulkner et al. stimulus frequency until the force reached a plateau. For 2008). Similar declines in the structure and function each of the two muscles, the maximum isometric tetanic of skeletal muscles with ageing have been reported for force (Po) was produced typically at between 150 and mice (Brooks & Faulkner, 1988), rats (Kanda et al. 1986) 200 Hz. Two minutes were allowed between contractions and humans (Lexell et al. 1988). Prior to the present to enable the muscle to recover. A trace of a maximum experiments, animals were housed in a pathogen-free isometric contraction of an anterior tibialis (ATB) muscle barrier-protected animal room in the Unit for Laboratory of a WT mouse is presented in Fig. 2A. Note that in the Animal Medicine, University of Michigan. The procedures absence of the ‘yoke-apparatus’,there is a more rapid rise in used in the present study were approved by the University tension, a well-defined plateau in maximum force, and the of Michigan Committee on the Use and Care of Animals attainment of a slightly higher maximum force (Fig. 2Aa and were conducted in accordance with the National compared with Fig. 2Ab). Institute of Health Guide for Care and Use of Laboratory Animals. The experiments and all treatments of the mice and rats comply with The Journal of Physiology’s policy on ‘Yoke apparatus’ animal experimentation (Drummond, 2009). For the measurement of the lateral transmission of force from activated fibres to the surface of the muscles, a small plastic ‘yoke-apparatus’ was designed and fabricated Experimental procedures (Fig. 1A and B). A number of ‘yokes’ of different internal In preparation for the measurement of the forces circumferences were cut on a laser-cutting apparatus, with developed by skeletal muscles, each mouse or rat was the smallest ‘yoke’ with internal diameters from 2 mm to anaesthetized with an initial intraperitoneal injection of 4 mm and the largest ‘yoke’ from 4 mm to 8 mm, The pentobarbital sodium (40 to 60 mg kg−1). Throughout an availability of ‘yokes’ of different sizes enabled, for a given experiment, supplemental dosages maintained a depth experiment, the selection of a ‘yoke’ of the appropriate of anaesthesia that prevented responses to tactile stimuli size. Four holes for the attachments of sutures were and a heated platform maintained core temperature at spaced equally around the ‘yoke’. After the measurement 37◦C. For each mouse or rat, an incision was made on of maximum isometric force was made, the hook on the the left leg longitudinally from the knee to the ankle distal tendon of the muscle that attached to the force trans- and a blunt dissection technique freed the muscle from ducer was removed and a ‘yoke’ of the appropriate size was the surrounding . For the adult WT and slipped onto the muscle such that the ‘yoke’ fit snugly over mdx mice, the distal tendon of the anterior tibialis (ATB) the belly of the muscle at a point equidistant from the two muscle was exposed and cut just proximal to the super- ends. Once positioned, silk 6–0 sutures attached the ‘yoke’ ior transverse ligament and silk 4–0 sutures were tied firmly to the epimysium of the muscle at each of four to the distal tendon as close as possible to the muscle points around the belly of the muscle. A separate set of without damageing muscle fibres. For the rats, a similar four sutures attached to the ‘yoke’ were brought together dissection was used to prepare the extensor digitorum with a single attachment to the force transducer (Fig. 1A longus (EDL) muscle. For each of the two muscles, the and B). The four anchor points on the ‘yoke’ attached distal tendon of the muscle was folded back to form a to a single hook on the force transducer provided the short loop and tied again. The mouse or rat was placed measurement of the force transmitted laterally (Fig. 1B). in a prone position on the platform of the apparatus Although the ‘yoke’ attachment withstood forces up to with the left leg fixed at both the ankle and knee. An ∼1800 mN for ATB muscles of mice and ∼2000 mN for ‘S’ hook was attached to the force transducer (BG-1000; EDL muscles of rats, tearing of the epimysium occurred at Kulite Semiconductor products, Leonia, NJ, USA) and higher forces and a maximum isometric force could not then hooked through the loop of the distal tendon of be measured laterally. For the ATB muscle of the mouse the muscle. The exposed muscle and tendon were kept and the EDL muscle of the rat, there is essentially no

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Table 1. Summary of the measurements made on EDL muscles of rats and ATB muscles of mice

Muscle Muscle Specific force Po before yoke Po after yoke Lateral transmission Groups mass (mg) length (mm) (kN m−2) attachment (mN) attachment (mN) of force (mN)

EDL muscles of rats Young (n = 12) 147 ± 431± 2 265 ± 15 2995 ± 365 2300 ± 340 1830 ± 230 Old (n = 5) 135 ± 927± 3 205 ± 10 2230 ± 190 1790 ± 150 1350 ± 160 Very old (n = 6) 143 ± 330± 1 180 ± 5 1885 ± 160 1570 ± 190 1030 ± 120 ATB muscles of mice Control (n = 6) 53 ± 1 14.2 ± 0.1 250 ± 15 1695 ± 130 1220 ± 150 1050 ± 100 mdx (n = 6) 83 ± 1 14.2 ± 0.7 200 ± 10 2140 ± 70 1470 ± 85 815 ± 70

Values are means ± 1 S.D. Po is the maximum isometric tetanic force, and specific force is the Po normalized for the total cross-sectional area of the muscle. variation in the lengths of the fibres throughout either published data on the effect of age on these muscles in mice muscle. For EDL muscles of the mouse, the fibre length (Brooks & Faulkner 1988) and of age on these muscles in (Lf )/muscle length (Lm) ratio is 0.51 ± 0.18 and for the rats (Carlson et al. 2002). ATB muscle 0.61 ± 0.04 (Burkholder et al. 1994). Our data on EDL muscles of young (Lf /Lm = 0.45), adult = = (Lf /Lm 0.44) and old (Lf /Lm 0.45) mice (Brooks & Paired measurements of forces transmitted Faulkner, 1988) and unpublished data on rats are in good longitudinally and laterally agreement with these values. The homogeneity in the lengths of the individual fibres and the lengths of these For a given experiment, the appropriate branch of the fibres equalling almost half the length of the muscle are deep peroneal nerve was identified that innervated either the major reasons for the extensive use of these muscles the ATB muscle of mice, or the EDL muscle of rats. The in studies of ageing, fatigability and contractility (Brooks branch of the peroneal nerve was severed to provide a & Faulkner, 1996; Renganathan et al. 1997; DelloRusso sufficient length for the stimulation of the whole nerve to et al. 2001; Li et al. 2006). For the ATB muscles of obtain the maximum longitudinal force generated by the mice and the EDL muscles of rats, there was a loss in whole muscle from the proximal to the distal tendon (see maximum isometric force measured longitudinally after Table 1 for values and Figs 2Aa–c for the force traces of the attachment of the ‘yoke’ apparatus to the muscle, the contractions). Note that prior to the attachment of the but the deficit was not significantly different between the ‘yoke’, the force trace for both the WT and mdx muscles WT and mdx mice, or among the three age groups of displayed an almost vertical rise in force with a sharp rats. Following the attachment of the ‘yoke’ apparatus, transition to the plateau at maximum force (Fig. 2Aa). the maximum isometric force decreased on the average In contrast, after the attachment of the ‘yoke’, the force by 30% for the muscles of WT and mdx mice and 20% trace for both WT and mdx muscles (Fig. 2Ab and c) for the muscles of the young, old and very old rats. displayed a much slower initial rise in tension followed Fortunately, the losses in force due to the attachment of by a secondary slower rise to maximum force identified as the ‘yoke’ were linearly related to the forces developed in when the force traces began to show a decline (Fig. 2Ab and response to the stimulation of different numbers of motor c). Despite considerable impact of the ‘yoke’ on the rate of units. Consequently, the relationship between lateral and development of force, the maximum longitudinal forces of longitudinal force was not affected by the ‘yoke’ (Figs 1, the EDL muscles of young, old and very old rats measured 2, and 3). We conclude that the decrease in the maximum after the attachment of the ‘yoke’ was decreased by 20% force following the attachment of the ‘yoke’ apparatus and that of the ATB muscles of the WT and mdx mice was was attributable to the suturing of the ‘yoke’ apparatus decreased by 30% (Table 1). Following the measurement of to the epimysium of the muscle that led to accidental the maximum force transmitted longitudinally, the distal damage to single fibres close to the periphery of the tendon was detached from the force transducer and the muscles. The values for maximum specific longitudinal ‘yoke’ was attached for the measurement of the forces forces of the ATB muscles of WT (250 ± 15 kN m−2, n = 6) transmitted laterally from the proximal tendon to the and mdx (200 ± 10 kN m−2, n = 6) mice and for EDL ‘yoke’ (Figs 1C,2B and 3). Progressive dissections of the muscles of young rats (265 ± 15 kN m−2, n = 12) with motor nerve produced a wide range of paired longitudinal an age-related decrease of 20% for the muscles old rats and lateral forces eventually down to one or two motor (205 ± 10 kN m−2, n = 5) and 30% for the muscles of units for ATB muscles of both WT and mdx mice (Figs 1C very old rats (180 ± 5kNm−2, n = 6) are consistent with and 2B) and for EDL muscles of young, old and very old

C 2011 The Authors. Journal compilation C 2011 The Physiological Society J Physiol 589.5 Lateral transmission of force in skeletal muscles of mice and rats 1199 rats (Fig. 3). When measurements of the paired forces were on 3–15% gradient SDS-PAGE and Western blotting on completed, the proximal tendon was severed to separate PVDF membranes. Equivalent loading was confirmed the muscle from the leg, the ‘yoke’ was removed from by Ponceau S staining of the transferred membrane. the muscle and the muscle was blotted and weighed. The Blots were blocked with 5% non-fat dry milk in TBS-T animal was then given an overdose of anaesthesia and a (50 mM Tris,pH7.5,150mM NaCl + 0.01% Tween bilateral pneumothorax performed to ensure killing. 20). Protein expression was determined using anti- bodies against dystrophin (Mandra, Sigma), glycosylated α-dystroglcyan (IIH6, Upstate Biotechnology/Millipore), Histological analysis β- (Santa Cruz Biotechnology), B1 integrin (Chemicon/Millipore) and (Hamlet, Vector Skeletal muscles were removed from anaesthetized rats Laboratories, Inc., Burlingame, CA, USA) followed and embedded fresh in a TFMTM tissue freezing medium by horseradish peroxidase-conjugated secondary anti- (Triangle Biomedical Science, Durham, NC, USA). They bodies (Jackson ImmunoResearch Laboratories, Inc., West were then snap frozen in liquid nitrogen-cooled iso- Grove, PA, USA) both diluted in blocking buffer and pentane. Transverse cryosections (8 μm) were cut through incubated for 1 h at room temperature. Blots were the middle of the muscle and were sectioned in a washed three times with TBS-T after each incubation. Leica CM3050 cryostat. For histological staining, frozen Blots were developed with ECL reagents (Pierce/Thermo sections were fixed in 10% neutral buffered formalin Scientific) according to manufacturer directions and and then stained with 0.1% picrosirus red/0.1% fast visualized and quantified using a Fluorchem imageing green for 30 min for the identification of the inter- sytem and AlphaView software for densitometry (Alpha stitial connective tissue. For immunohistological staining, Innotech/Cell Biosciences, Santa Clara, CA, USA). To sections were blocked with 5% BSA in phosphate buffered normalize the changes in protein expression, the data saline (PBS) for 1 h and stained with primary anti- obtained on densitometry of the skeletal muscles of old bodies to IV (Santa Cruz Biotechnology, Inc., and very old rats were divided by the mean of the Santa Cruz, CA, USA) and pan-laminin (Sigma-Aldrich expression levels in the young rats to demonstrate the Corp., St Louis, MO, USA) followed by Cy3 conjugated relative magnitude of the change in protein expression secondary antibodies. All antibodies were diluted in with age. blocking solution, and slides were washed in three washes of PBS between incubation. Following the staining procedure, the coverslips were mounted on the slides with Results Permafluor (Thermo Scientific). Slides were viewed and captured on an Olympus BX51 fluorescence microscope Lateral transmission of force in skeletal muscles with a DP-71 digital camera. Images were analysed by of young, wild-type mice and rats ImageJ software to measure cross sectional areas (available The measurements of paired values for lateral and at http://rsbweb.nih.gov/ij/). longitudinal forces developed by the muscles of young WT mice (shown in Fig. 1C), young WT and mdx mice (shown in Fig. 2C) and young, old and very old rats Protein expression (shown in Fig. 3) allowed comparisons from maximum KCl washed microsomes were prepared from homo- whole muscle force to that of just a few motor units. The genates of hind-limb muscles from young and very old low forces, elicited by activating a small number of motor rats. Briefly, muscles were homogenized using a dounce units in WT muscles of mice and rats, were transmitted homogenizer in buffer containing (in mM): 20 sodium equally well laterally and longitudinally and, even with pyrophosphate, 20 sodium phosphate, 1 MgCl2, 300 high levels of activation, the forces at the ‘yoke’ sucrose, 5 EDTA, pH 7.1. Homogenates were centrifuged averaged only slightly less than those measured at the distal for 25 min at 14,000 g. The supernatant was collected and tendon. For the WT muscles of both species, the small centrifuged for 35 min at 30,100 g. The pellet was then loss in the lateral, compared with the longitudinal, trans- resuspended and incubated in buffer containing 50 mM mission of force observed at the highest forces generated ◦ Tris,pH7.4,0.3M sucrose, 0.6 M KCl for 30 min at 4 C was attributed to: (i) the compliance of the attachments of with gentle stirring. The resultant KCl washed micro- the ‘yoke’ to the surface of the muscle and (ii) a gradient somes were then collected by centrifugation at 140,000 g in the amount of shortening that occurred in the lengths for 30 min and resuspended in 50 mM Tris,pH7.4,0.3M of fibres between the fibres at the periphery and those at sucrose. All buffers contained a combination protease the core of the muscles with the fibres in the core of the inhibitor cocktail and were used ice cold. Membrane muscle shortening slightly more than those nearer to the protein concentration was determined by the Lowry ‘yoke’ (Fig. 1D). Both of these factors resulted in some assay and equivalent amounts of protein were loaded fibres shortening to lengths that were less than optimum

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Figure 1. The apparatus used to measure the longitudinal and lateral transmission of force A, transmission of the force was measured from the proximal to the distal tendon to assess the longitudinal transmission of force. B, transmission of the force was measured from the proximal tendon laterally from the isometrically contracting fibres to the yoke. C, longitudinal and lateral force transmission in WT anterior tibialis anterior muscle. Note the closeness of the data to the line of identity between lateral and longitudinal force.

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Figure 2. Comparison of the longitudinal and lateral transmission of forces in WT and mdx mice Aa, a record of the maximum isometric tetanic force (Po) of an ATB muscle of a WT mouse. Note ATB and EDL muscles of WT and mdx mice and young and old WT rats display a very similar type of isometric force trace. Ab and c, records obtained following the attachment of the ‘yoke’ apparatus. The forces were measured during maximum stimulation of the motor nerve with the muscle at optimum length for the production of force (Lo). Following the attachment of the ‘yoke’ apparatus, the longitudinal and lateral force traces of maximally activated WT (b)andmdx (c) muscles of mice displayed a slower initial rise in force and then a secondary even slower rise. After the attachment of the ‘yoke’, the Po valuesoftheWTandmdx muscles were ∼25% lower than those of the muscles before the attachment of the ‘yoke’. Note the much closer relationship for the values for the lateral and longitudinal forces for the WT muscle than for the mdx muscle. B, relationship of longitudinal to lateral force transmission in anterior tibialis (ATB) muscles of mdx and WT mice.

for the development of maximum force (Gordon et al. Lateral transmission of force is impaired 1966). Despite these factors and the substantially higher in dystrophin-deficient mdx mice forces developed in the experiments reported here, the results are in excellent agreement with the single fibre Given that null mutations in the dystrophin gene lead to data obtained on the muscles of frogs (Street, 1983). We a loss of dystrophin expression in the sarcolemma of mdx conclude that, as demonstrated previously in the muscles muscles, we tested the hypothesis that the lateral trans- of frogs, the forces developed by WT muscles of young mission of force is impaired significantly in mdx compared mice and rats are transmitted equally well laterally and with WT muscles. In contrast to the close approximation longitudinally. to the line of identity for the forces generated by the

D, diagram showing a cross-section of a skeletal muscle during the measurement of lateral transmission of forces. Note that throughout the contraction the fibres close to the surface of the muscle remain isometric between the proximal tendon and the yoke at an optimum length for force development. In contrast, the fibres toward the center of the muscle cross-section tend to shorten slightly and contribute less force than the fibres at optimum length.

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WT muscles and transmitted laterally to the ‘yoke’, those ability of mammals to generate force (Young et al. 1984, generated by the mdx muscles and transmitted laterally 1985; Brooks & Faulkner, 1988; Faulkner et al. 2008) to the ‘yoke’ diverged at even the lowest forces measured. and power (Faulkner et al. 2008). Due to the inability For the data plotted, the difference between the slopes of of old muscles and very old muscles to produce the same the best-fit lines for each data set (0.46 for the mdx and level of force as young muscles, the comparison of the 0.78 for the WT muscles) indicate clearly a much closer lateral transmission of forces between age groups was relationship between the force measured at the ‘yoke’ and achieved by taking the ratio of lateral to longitudinal the force measured at the distal tendon for the WT than transmissions. Figure 3 shows an age-related difference in for the mdx muscles. For the WT muscles, the forces the lateral transmission of force within the EDL muscles transmitted laterally and longitudinally did not diverge among the three age groups, young, old and very old. significantly from the line of identity until force levels The difference in the lateral transmission of force between exceeded 900 mN, a value ∼50% of the maximum iso- the old and the very old (P ≤ 0.001) muscles is much metric force developed by the WT muscles. The magnitude greater than the difference between young and old muscles of the differences between the lateral compared with the (P > 0.05), even though the difference in age between the longitudinal transmission of force generated by the mdx young and old muscles is greater than that between the compared with the WT muscles was greatest at the highest old and very old muscles. The deficit in the lateral trans- forces measured, with a deficit of ∼50% for the mdx mission of forces for both old and very old muscles was compared with ∼20% for the WT muscles. The impaired particularly noticeable at very high forces, greater than capability of the mdx muscles to transmit force laterally 1000 mN. For forces less than 1000 mN, the young and provides a direct demonstration that the DGC is a major old muscles expressed similar levels for the lateral trans- factor in the lateral transmission of force and provides mission of force, whereas very old muscles expressed much strong support for the models proposed for the lateral lower values for the lateral transmission of force at all levels transmission of force in mammalian muscle that were of force production. until now hypothetical (Pardo et al. 1983; Hoffman et al. 1987; Ervasti & Campbell, 1993; Worton, 1995; Rybakova et al. 2000; Paul et al. 2002; Bloch & Gonzalez-Serratos, Alterations in the interactions of muscle fibres 2003; Ervasti, 2003; Huijing, 2003; Michele & Campbell, with ECM within muscles of very old rats 2003; Bloch et al. 2004; Lapidos et al. 2004; Anastasi et al. 2008; Claflin & Brooks, 2008). Many of the molecular components responsible for the lateral transmission of force in skeletal muscles are unknown particularly those at the interface between Lateral transmission of force is impaired with ageing the costameres and the ECM (Rybakova et al. 2000; As with muscular dystrophy, advanced age leads to major Bloch & Gonzalez-Serratos, 2003; Ervasti, 2003; Lapidos decrements in muscle function that include a loss in the et al. 2004). Despite the lack of knowledge regarding the specific components, lateral transmission of force involves proteins that link the costameres functionally to the ECM as well as the actual composition of the ECM (Kjaer, 2004; Gao et al. 2008a,b). To test whether these components were altered in old and very old muscles, expression levels of components of the DGC and the integrin complex, key costameric ECM receptor complexes in skeletal muscle, were compared in membranes isolated from young (6 months old) and very old (35–36 months old) muscles. The expression of dystrophin was reduced by more than 60% in the membrane fractions of very old compared with young muscles (Fig. 4A and B). This large change in dystrophin protein expression was quite unique, as expression levels of other components of the DGC, including β-dystroglycan and α-dystroglycan, were not changed significantly (Fig. 4A and B). Conversely, the Figure 3. Lateral and longitudinal transmission of force for expression levels of β1 integrin were increased significantly EDL muscles of young, old and very old rats in very old muscles (Fig. 4A and B). Values are given as the maximum isometric tetanic forces measured To determine the specificity of the decrease in from proximal to distal tendon (longitudinal force) and from the yoke to the proximal tendon (lateral force) in cases of whole muscles dystrophin expression in very old muscles, the expression as well as those from decreased sets of motor units. levels of dysferlin, a membrane protein important in

C 2011 The Authors. Journal compilation C 2011 The Physiological Society J Physiol 589.5 Lateral transmission of force in skeletal muscles of mice and rats 1203 muscle vesicle trafficking and repair of membrane damage lamina was especially noticeable in the area of contact (Bansal et al. 2003), was examined. Similar to the null amongst three neighbouring muscle fibres (Fig. 5A). In mutations in dystrophin, null mutations in dysferlin can very old muscles, capillaries located in this area were cause muscular dystrophy, even though dysferlin is not completely surrounded by a thick basal lamina. To an integral component of the DGC (Bansal et al. 2003). quantify collagen deposition in very old muscles, muscle Although in isolated membranes from very old muscles, sections were stained with picrosirius red and fibrillar the expression levels of dysferlin were normal (Fig. 4C), collagen content was quantified by ImageJ analysis. with immunolocalization, the dysferlin expressed was (Fig. 5B and C). The very old muscles showed a more redistributed to membranes within the cytoplasm than 5-fold increase in fibrillar collagen expression. (Fig. 4D). In several forms of muscular dystrophy that are Immunolocalization techniques were used to measure associated with null mutations in components of the DGC, the expression of key molecular components of the basal dysferlin is redistributed secondarily from the sarcolemma lamina. The components included laminin and type IV to membrane compartments within the cytoplasm. collagen indicating that some key components were In very old muscles that showed reduced dystrophin preserved in the very old muscles (Fig. 5B). The apparent expression, no up-regulation of the dystrophin homologue thickness of the staining for each of these components utrophin was observed (Fig. 4E). Consequently, for very was increased, which was consistent with our observations old muscles, the loss of dystrophin expression was not made with electron microscopy. Furthermore, in very compensated for by an increased expression of utrophin. old muscles, QRT-PCR showed no detectable increase in This placed the very old rats in a similar situation to that collagen IV, procollagen I and laminin mRNA expression of dystrophic mice, with skeletal muscles that are seriously (data not shown). These observations suggest that during impaired in their ability to transmit force laterally. the ageing of skeletal muscles, the accumulation of the Very old muscles also show significant remodelling components of the basal lamina and the connective tissue of the ECM (Kjaer, 2004; Gao et al. 2008a,b). Electron was likely associated with a decreased degradation of micrographs of very old muscles showed a significant the ECM proteins. In summary, while alterations were increase in the thickness of the basal lamina surrounding observed in the deposition of the ECM in very old the muscle fibres (Fig. 5A). The thickening of the basal muscles, the molecular composition of the basal lamina

Figure 4. Expression of dystrophin– glycoprotein complex and integrin complex proteins in aged muscle membranes One hundred micrograms of total hindlimb muscle membrane protein preparations from three young (12 month) and three very old rats (34–35 months) was analysed by SDS-PAGE and Western blotting (A) and quantified by densitometry (B) ∗P < 0.05 by Student’s t test, data are means ± S.E.M. C and D, mislocalization of dysferlin and no upregulation of utrophin in aged muscle. While expression levels of dysferlin in membrane fractions is normal by Western blotting (C), immunolocalization of dysferlin (D) shows redistribution of dysferlin expression into cytoplasmic membrane compartments. E,no upregulation of utrophin was observed in aged muscle, although full length utrophin protein was detected in a positive control sample from neonatal muscle.

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and matrix appeared to be preserved. Furthermore, the integrin mediated linkages appeared to be preserved, or even increased. These observations, combined with our results that the loss of dystrophin was sufficient to cause a decrease in the transmission of the lateral force, shift the emphasis for the age-related decline in the lateral transmission of force squarely onto an acquired loss of dystrophin expression.

Discussion

The experiment performed by Street (1983) demonstrated for the first time that when a single fibre partially dissected from the semitendinosis muscle of a frog was activated maximally, the force was transmitted laterally across the adjacent fibres to the epimysium with little or no decrement. The skeletal muscles of frogs have a much simpler architecture and design than mammalian skeletal muscles (Patel & Lieber, 1997; Huijing, 2003), but the transverse lattice elements (‘costameres’) were already under investigation in mammals (Pardo et al. 1983) and potential roles for the dystrophin–glycoprotein complex (Ervasti & Campbell, 1993; Paul et al. 2002; Michele & Campbell, 2003; Lapidos et al. 2004) in the lateral trans- mission of force were quite evident. We now demonstrate in skeletal muscles of WT mice and rats that forces generated during activation of either the whole muscle or of varying numbers of motor units are transmitted laterally to the epimysium with little or no decrement. In contrast, in the skeletal muscles of mdx mice and very old rats the lateral transmission of force was impaired severely. In each case, the magnitude of the impairment in the lateral transmission of force was correlated closely with a loss of dystrophin expression. Our data indicate that either the loss of dystrophin in mdx mice, or the acquired disruption of the DGC, as a consequence of the ageing of WT rats, leads to significant losses in the capability of skeletal muscle fibres to transmit forces laterally. Within single fibres, the stability of the sarcomeres is highly dependent on shear linkages of sarcomeres to

muscles of young (left) and old (right) rats are shown. Bar = 2 μm. B, increased abundance of fibrillar collagen and thickness of collagen IV and laminin in the basal lamina of muscle from very old rats. Picrosirius red staining followed by area analysis was used to quantify the fibrillar collagen expression in aged muscle. Immunolocalization of type IV collagen and laminin show increased thickness and staining intensity of the basal lamina. Bar = 200 μm, top two panels. Bar = 100 μm, immunofluorescence panels. Figure 5. Increase in accumulation of interstitial connective Magnification of the bottom panels was increased identically for tissue and basal lamina thickness in skeletal muscles of old detailed comparative purposes. C, collagen composition. Two entire rats midbelly sections of picrosirius red stained sections from each muscle A, electron micrographs of three adjacent fibres of an EDL muscle were quantified using a montage of 10× objective images, and showing thickening of the basal lamina in skeletal muscles of old averaged for each animal. ∗P < 0.005 by Student’s t test, data are compared to young rats. Three adjacent fibres from skeletal means ± S.E.M., n = 3 animals in each.

C 2011 The Authors. Journal compilation C 2011 The Physiological Society J Physiol 589.5 Lateral transmission of force in skeletal muscles of mice and rats 1205 the ECM (Huijing, 2003). The DGC, first described by Bloch & Gonzalez-Serratos, 2003; Ervasti, 2003; Huijing, Ervasti et al. (1990), resides within complex intracellular 2003; Bloch et al. 2004; Kjaer, 2004). During lengthening structures termed costameres (Pardo et al. 1983). The contractions in which high forces are developed within costameres are positioned in register with the z-discs of the fibres, the weaker groups of sarcomeres are at great risk myofibrils located on the periphery of each fibre (Pardo of being stretched beyond overlap of thick and thin et al. 1983; Ervasti & Campbell, 1991; Worton, 1995; filaments and of sustaining a contraction-induced injury Henry & Campbell, 1996; Bloch & Gonzalez-Serratos, (Macpherson et al. 1997; Panchangam et al. 2008). When 2003; Ervasti, 2003; Michele & Campbell, 2003; Bloch no mechanism is present for the lateral support from et al. 2004; Lapidos et al. 2004; Anastasi et al. 2008). adjacent fibres, as in dystrophic muscles, any variability Consequently, costameres are ideally positioned to trans- in development of forces along the lengths of fibres leads mit the forces developed by activated fibres laterally to heterogeneities in sarcomere lengths and to additional throughout a muscle. During each of the three types sarcomere injury (Macpherson et al. 1997; Claflin & of contractions, shortening, isometric and lengthening Brooks, 2008; Panchangam et al. 2008). Consequently, (Faulkner, 2003; Claflin & Brooks, 2008), the lateral trans- the lateral transmission of force appears to be critical mission of force stabilizes sarcomere lengths and forces both in the protection of fibres from sustaining the initial from fibre to fibre eventually out to the epimysium, as contraction-induced damage and for the healing of the demonstrated by Street (1983) in muscles of frogs, and damage after a contraction-induced injury (Dedrick & now by us in muscles of mice and rats. These lateral Clarkson, 1990; DelloRusso et al. 2001; Ploutz-Snyder et al. linkages are critical during each type of contraction, but 2001). Consequently, lateral pathways of support provide most critical during lengthening contractions, when forces the only mechanism that enables the longitudinal strains may be as much as twofold greater than those developed to be shunted around the injured segment of a fibre while it during isometric contractions (DelloRusso et al. 2001; is healing (Rader et al. 2006). The parallels between the loss Faulkner, 2003; Li et al. 2006). Our results on the very old of dystrophin, the disruption of the DGC, the decrease in musclesofratsshowforthefirsttimethatanage-related the lateral transmission of force and the greatly increased loss of dystrophin is sufficient to cause a marked loss in susceptibility to contraction-induced injury in the skeletal the lateral transmission of force, which provides important muscles of both dystrophic mice and humans and in experimental evidence that the DGC and costameres play very old mammals provide strong support for the inter- critical roles in lateral transmission of force in skeletal dependence of these variables and their vital importance in muscles. the normal maintenance of the viability of skeletal muscle The possibility that dystrophin plays an important fibres. role in the stability of skeletal muscle fibres and its Descriptive changes of the losses in the structure and absence in dystrophic muscles contributes to the fragility function of ageing skeletal muscles are well-documented of dystrophic muscle has been proposed by a number in humans (Grimby & Saltin, 1983; Young et al. 1984, 1985; of investigators (Ervasti et al. 1990; Worton, 1995; Faulkner et al. 2008), rats (Larsson et al. 1991) and mice Henry & Campbell, 1996; Ervasti, 2003; Michele & (Brooks & Faulkner, 1988). These losses are explained, Campbell, 2003; Lapidos et al. 2004). Following protocols at least in part, by the linear decrease in the number of of lengthening contractions, the skeletal muscles of mdx fibres in skeletal muscles of humans that begins in the mice (DelloRusso et al. 2001; Li et al. 2006) and old mid-fifties (Lexell et al. 1988) and is coupled with the rats (Brooks & Faulkner, 1996) and humans (Dedrick timing and linear losses in the number of motor neurons & Clarkson, 1990; Frontera et al. 1991; Ploutz-Snyder (Lexell et al. 1988) and consequently in the number of et al. 2001) exhibit high levels of contraction induced motor units (Campbell et al. 1973; Doherty et al. 1993). injury. Compared with the force deficits elicited by a Studies of alterations in excitation–contraction coupling lengthening contraction protocol administered to skeletal have clarified, at least in part, the impaired production muscles of young WT mice, force deficits for mdx mice of muscle force by the skeletal muscles of old animals were sevenfold greater (DelloRusso et al. 2001; Li et al. (Renganathan et al. 1997), but many issues that contribute 2006) and for those of old compared with young rats to the decreased force production and the susceptibility of twofold greater (Brooks & Faulkner, 1996). The large the skeletal muscles of old and very old animals to atrophy force deficits sustained by dystrophin-deficient muscles and to injury remain unresolved. Consequently, although of mdx mice, even during isometric contraction protocols the gross whole skeletal muscle changes that occur with (Claflin & Brooks, 2008), reflect the compromised lateral ageing are well-described (Faulkner et al. 2008), the under- transfer of forces between activated fibres, as each fibre lying molecular bases for many of these structural and acts as an independent longitudinal force generator. functional changes that occur in skeletal muscles and These observations are strongly supportive of the critical single fibres with ageing are complex and largely unknown. role that has been proposed for the costameres in the Our observation that the lateral transmission of force maintenance of sarcomere stability (Rybakova et al. 2000; is disrupted in the skeletal muscles of mdx mice and old

C 2011 The Authors. Journal compilation C 2011 The Physiological Society 1206 K. S. Ramaswamy and others J Physiol 589.5 and very old rats focuses attention on molecules involved compared with the longitudinal transmission of force, the in the interactions between the DGC in muscle fibres and lateral transmission of force is affected disproportionately. the ECM (Kjaer, 2004; Gao et al. 2008a,b)aspotential The hypertrophy of the EDL and soleus muscles of mdx causes of some of the disease and age-related dysfunctions. compared with WT mice has been reported previously Costameric proteins, including β1-containing integrins (Lynch et al. 2001). The hypertrophy of the mdx muscles and the DGC, play critical roles in linking sarcomeres to results in the mdx muscles developing maximum forces the basal lamina and subsequently to the ECM (Worton, longitudinally that are not different from those developed 1995; Rybakova et al. 2000; Paul et al. 2002; Ervasti, 2003; longitudinally by the WT muscles (Lynch et al. 2001). Lapidos et al. 2004). In the muscles of very old rats, Despite the lack of any difference in the longitudinal the loss of lateral transmission of force was associated forces developed by mdx and WT muscles, the lateral with a significant loss of dystrophin expression, while forces developed by the mdx muscles are compromised integrin and ECM protein expression was increased. Inter- severely. The approaches developed in this study caused estingly, when extrapolated to maximum isometric force, deficits in the lateral transmission of force in animal the magnitude of the loss in the lateral transmission of model systems that produced both morphological and force observed for the muscles of very old rats (30%) was physiological changes, primarily through the disruption only slightly more than half the magnitude of the loss of the DGC. In future studies, such approaches offer associated with the total loss of dystrophin in the young opportunities to identify the intracellular and extracellular mdx mice of (50%). These values correlate reasonably molecular components of the longitudinal and lateral well with the 60% loss in dystrophin expression in very transmission of force in dystrophic and very old skeletal old rats compared with the 100% loss of dystrophin muscles, as well as their contributions to other forms of expression in the mdx mice. Consequently, the acquired muscle dysfunction. loss of dystrophin expression in the skeletal muscles of very old rats has the potential of not only playing a critical role in the impairment in the lateral transmission of References force, but also being involved in the broader, whole-body characteristics of ageing, such as frailty, fatigability and Abmayr S & Chamberlain JS (2006). The structure and function susceptibility to injury (Pirozzolo & Maletta, 1982; Grimby of dystrophin. In The Molecular Mechanisms of Muscular Dystrophy, ed. Winder S, Landes Bioscience, Georgetown. & Saltin, 1983; Faulkner et al. 2008). Anastasi G, Cutroneo G, Santoro G, Arco A, Rizzo G, Bramanti We propose that both dystrophin and the DGC P, Rinaldi C, Sidoti A, Amato A & Favaloro A (2008). play critical roles in the transmission of the lateral Costameric proteins in human skeletal muscle during force in skeletal muscles, although the possibility exists muscular inactivity. J Anat 213, 284–295. that other secondary morphological changes in mdx Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, muscles in mice and/or very old muscles in rats might McNeil PL & Campbell KP (2003). Defective membrane contribute to the observed deficits in lateral force. repair in dysferlin-deficient muscular dystrophy. Nature 423, While even very old muscles do not show cycles of 168–172. degeneration/regeneration, both older dystrophic muscles Bloch RJ & Gonzalez-Serratos H (2003). Lateral force in mice (Pastoret & Sebille, 1995; Head, 2010) and very transmission across costameres in skeletal muscle. Exerc old muscles in rats show changes in the ECM that include Sport Sci Rev 31, 73–78. Bloch RJ, Reed P, O’Neill A, Strong J, Williams M, Porter N & fibrosis, increased variation in fibre size (Fig. 5), fibre Gonzalez-Serratos H (2004). Costameres mediate force atrophy and the occasional splitting of muscle fibres (see transduction in healthy skeletal muscle and are altered in online Supplemental Material, Supplementary Fig. 1). The muscular dystrophies. JMuscleResCellMotil25, possibility exists that for the skeletal muscles of both 590–592. very old WT rats and mdx mice, the ECM, including Brooks SV & Faulkner JA (1988). Contractile properties of the epimysium, becomes stiffer due to fibrosis (Kjaer, skeletal muscles from young, adult and aged mice. JPhysiol 2004; Gao et al. 2008a,b). This possibility is supported by 404, 71–82. the similarities in the rates and the eventual magnitude Brooks SV & Faulkner JA (1996). The magnitude of the initial in the loss in the development of lateral force – 30% injury induced by stretches of maximally activated muscle for the very old muscles of rats and 50% for the mdx fibres of mice and rats increases in old age. JPhysiol497, muscles of mice. This observation suggests that the over- 573–580. Burkholder TJ, Fingado B, Baron S & Lieber RL (1994). all compliance of the skeletal muscles in each of these two Relationship between muscle fiber types and sizes and mammalian species, is similar. Many of the morphological muscle architectural properties in the mouse hindlimb. changes observed for the dystrophic and very old muscles JMorphol221, 177–190. contribute to defects in the longitudinal as well as the Campbell MJ, McComas AJ & Petito F (1973). Physiological lateral transmission of force. In that respect, an extremely changes in aging muscles. J Neurol Neurosurg Psych 36, interesting observation is that for each of these two models, 174–182.

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Paul AC, Sheard PW, Kaufman SJ & Duxson MJ (2002). Young A, Stokes M & Crowe M (1985). The size and strength of Localization of α7 integrins and dystrophin suggests the quadriceps muscles of old and young men. Clin Physiol 5, potential for both lateral and longitudinal transmission of 145–154. tension in large mammalian muscles. Cell Tissue Res 308, 255–265. Author contributions Pirozzolo FJ & Maletta GJ (1982). The Aging Motor System. Praeger Publishers, New York. The work was performed in the research laboratories of D.E.M. in Ploutz-Snyder LL, Giamis EL, Formikell M & Rosenbaum AE the Department of Molecular & Integrative Physiology and J.A.F. (2001). Resistance training reduces susceptibility to eccentric in the Biogerontology Laboratories in the Biomedical Sciences exercise-induced muscle dysfunction in older women. Research Building at the University of Michigan Medical School. JGerontolABiolSciMedSci56, B384–B390. K.S.R, D.E.M. and J.A.F. contributed to the conception and Rader EP, Song W, Van Remmen H, Richardson A & Faulkner design of the experiments. K.S.R., M.L.P., J.M.VM., A.R., T.Y.K., JA (2006). Raising the antioxidant levels within mouse D.E.M. and J.A.F. contributed to the overall collection, analysis muscle fibres does not affect contraction-induced injury. and interpretation of the data. K.S.R, D.E.M. and J.A.F. drafted Exp Physiol 91, 781–789. the article and revised it critically for intellectual content. All of Renganathan M, Messi ML & Delbono O (1997). the authors approved of the final version of the manuscript. Dihydropyridine receptor-ryanodine receptor uncoupling in aged skeletal muscle. J Membr Biol 157, 247–253. Rybakova IN, Patel JR & Ervasti JM (2000). The dystrophin Acknowledgements complex forms a mechanically strong link between the sarcolemma and costameric actin. J Cell Biol 150, 1209–1214. We thank Robert G. Dennis for assistance in the design and Street SF (1983). Lateral transmission of tension in frog fabrication of the yoke apparatus, Dennis R. Claflin for his myofibers: a myofibrillar network and transverse cytoskeletal helpful comments on the manuscript, Cheryl Hassett of the connections are possible transmitters. JCellPhysiol114, Faulkner Laboratory for methodological assistance to K.R. with 346–364. the operations and collection of data. Carol S. Davis assisted in Worton R (1995). Muscular dystrophies: diseases of the the preparation of the manuscript for publication. This work dystrophin-glycoprotein complex. Science 270, 755–756. was supported by funds to J.A.F. from the Contractility Core of Young A, Stokes M & Crowe M (1984). Size and strength of the the Nathan Shock Centre P30 AG13283 and the Functionality quadriceps muscles of old and young women. Eur J Clin Core of the Program Project PO1 AG15434 and to D.E.M. from Invest 14, 282–287. NIH R01-HL080388.

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